Previous research suggests that even in turbulent flow the rate of O2 uptake by human erythrocytes is partly limited by an extracellular diffusion boundary layer (or unstirred layer). Among the specific aims of this project are: (1) to obtain more direct evidence for the presence or absence of the diffusion boundary layer, (2) to quantify the O2 permeabilities of diffusion boundary layer and erythrocyte plasma membrane, (3) to determine the influence of physical properties of the red cell on diffusion boundary layer, (4) to determine the influence of increased membrane cholesterol and anesthetic alcohols on the O2 permeability of the red cell membrane, and (5) to determine the effects of emulsified fluorocarbon on the O2 permeability of the diffusion boundary layer. To accomplish our objectives we will use erythrocytes labeled at their external surfaces with pyrene -- an O2-quenched fluorophore. Monitoring the pyrene fluorescence in a stopped-flow apparatus will allow us to determine the PO2 at the erythrocyte membrane surface during the time course of O2 uptake. Knowing the PO2 at the membrane surface and the rate of O2 uptake, we will calculate the O2 permeabilities of the diffusion boundary layer and the red cell membrane under the conditions mentioned. The long-term objectives of our research are to define the factors that limit the rate of gas exchange by human erythrocytes in vivo. In the research described in this proposal we will define the factors that limit O2 uptake in a stopped-flow rapid reaction apparatus under defined conditions of flow. When we understand the factors that limit gas exchange by erythrocytes in this relatively simple system, we will be in a better position to ask what factors limit gas exchange by erythrocytes in vivo in the capillaries that supply the pulmonary alveoli and the peripheral tissues. The scientific disciplines involved in our work are: physiology, biophysics, and biochemistry. When the factors that limit the rate of gas exchange by erythrocytes in vivo are better understood, this knowledge may be useful in devising improved therapies for individuals who suffer from diseases that involve impaired gas exchange by erythrocytes.